NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
HIV Infection HIV Infection
Laboratory diagnosis and Laboratory diagnosis and monitoring 2006monitoring 2006
Philip Cunningham
NSW State Reference Laboratory for HIV/AIDS
St Vincent’s Hospital Sydney
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
HIV laboratory testsHIV laboratory tests
diagnosisdiagnosis of infectionof infectionacute, recent, established or late stage disease
monitoringmonitoring of ARV therapiesof ARV therapiesimmunological and virological markerspharmacologytoxicities surrogate prognostic markershost factors
diagnosis of opportunistic infectionsdiagnosis of opportunistic infections
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
‘‘typicaltypical’’ primary HIVprimary HIV--1 infection1 infection
symptoms
HIV-1 p24 antigen
0 1 2 3 4 5 6 / 2 4 6 8 10weeks yearsTime following infection
HIV viral load
HIV proviral DNA
symptoms
‘‘windowwindow’’periodperiod
1° infection
HIV antibodies
Limit of detection
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
HIV Testing HIV Testing Virus Specific AntibodiesVirus Specific Antibodies
Screening test Screening test –– ELISA, EIA, particle agglutinationELISA, EIA, particle agglutinationReference (confirmatory) testing Reference (confirmatory) testing –– 22ndnd EIA, western blotEIA, western blotSupplemental testing Supplemental testing –– resolve discordant testsresolve discordant testsSerum rapid testsSerum rapid testsSurveillance testing Surveillance testing –– monitoring incidencemonitoring incidence
Testing strategy dictated by populationTesting strategy dictated by populationsensitivity sensitivity vsvs specificityspecificity
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
ELISA ELISA –– EEnzyme nzyme LLinked inked IImmunosorbent mmunosorbent AAssayssay
detect HIVdetect HIV--1 and HIV1 and HIV--2 responses2 responsesHIV specific antibody and HIV antigen may be HIV specific antibody and HIV antigen may be detected in detected in ‘‘44thth’’ generation testsgeneration testssensitive, specific and reproduciblesensitive, specific and reproduciblefalse reactions possible false reactions possible –– but lowbut lowsuitable for large number of tests suitable for large number of tests –– automatedautomatedMedicare listed November 2005Medicare listed November 2005
Screening widely availableScreening widely availableConfirmation in reference laboratoriesConfirmation in reference laboratories
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Antibody/antigen comboAntibody/antigen combo
HIVHIV--1/2 Ag/1/2 Ag/AbAb combo assays detect both antibodies (HIVcombo assays detect both antibodies (HIV--1+2) 1+2) and viral antigen (HIV p24) in single testand viral antigen (HIV p24) in single testResult in 60 minutesResult in 60 minutes
Reduction in window period by 3Reduction in window period by 3--5 days5 daysAcute infection detection without indicationReports of increases in cases identified
Differences in limit of detection of Ag between brandsDifferences in limit of detection of Ag between brands(140 - <25 pg/mL)
Issues associated with introductionIssues associated with introductionStrategies and confirmatory algorithmsCost and legal
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
HIV HIV AbAb/Ag Combo strategy/Ag Combo strategyHIV-1/2 Ab/Ag combo
(x1)
Non reactive (Ag/Ab) Reactive (Ab/Ag)
Negative Non-reactive (Ab/Ag)(x2)
Repeat-reactive (Ab/Ag)(x2)
Negative
HIV-1 western blot (Ab)
Genescreen (Ab only) (x2)
Positive Negative/Indeterminate
Additional Tests HIV-1 p24 antigen
DNA PCR HIV-2
Negative
Indeterminate
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Serology of primary Serology of primary HIVHIV--1 infection1 infection
Antibody responses to Antibody responses to immunogenic proteins immunogenic proteins occur in typical series occur in typical series -- diagnosticdiagnostic
All viral proteins are All viral proteins are detected detected –– increasing increasing in intensityin intensity
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Effect of antiretroviral Effect of antiretroviral therapytherapy
Antigenic stimulus is Antigenic stimulus is reducedreduced
EnvEnvelope responses elope responses preserved preserved –– high antigenic high antigenic diversity to glycoprotein diversity to glycoprotein --less redundancyless redundancy
Responses to Responses to gaggag discrete discrete structural proteins are lost structural proteins are lost -- redundancy redundancy
DonDon’’t reach positive status t reach positive status –– underreporting underreporting
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Advanced HIV infectionAdvanced HIV infection
Low level antibody test Low level antibody test reactivity reactivity Indeterminate profile Indeterminate profile ––pattern typical of late stagepattern typical of late stageRetain glycoprotein Retain glycoprotein reactivityreactivityLate presentersLate presenters
helpfulhelpfulCD4 lymphocyte countCD4 lymphocyte countHIV HIV proviralproviral DNA PCRDNA PCRHIV viral loadHIV viral load
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
DNA PCRRNA PCR
p24 Ag3rd gen ELISA1st gen ELISA
Detuned ELISA
1wk 2wk 3wk 2mo 6mo 1yr 2yr 3yr +8yr
gp160gp120
p68p55p53
gp41-45
p40
p34
p24
p18
p12
gp160gp120
p68p55p53
gp41-45
p40
p34
p24
p18
p12
gp160gp120
p68p55p53
gp41-45
p40
p34
p24
p18
p12
acute established late
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Window periodWindow period
antibodies appear within 3antibodies appear within 3--4 weeks4 weeks
Antibody testing strategies have limitations Antibody testing strategies have limitations –– 100% 100% people seroconvert within 12 weeks people seroconvert within 12 weeks
More sensitive testing strategies including direct More sensitive testing strategies including direct detection of virus (NAT) and 3detection of virus (NAT) and 3rdrd / 4/ 4thth generation generation immunoassays will reduce window period to 6 immunoassays will reduce window period to 6 weeksweeks
HAART therapy during primary infection = delayed HAART therapy during primary infection = delayed antibody responseantibody response
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
HIV TestingHIV TestingDirect Detection of VirusDirect Detection of Virus
p24 antigen detection – serologyp24 only assays – qualitative and quantitativep24 in combination with antibody Serum
Virus isolation - cultureNucleic acid detection - (NAT))
HIV DNA or RNA ?DNA qualitative – proviral (cellular)
resolution of inconclusive serologydiagnosis in infants - maternal antibodies
RNA quantitative – serial viral loaddrug resistance monitoring subtyping
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Pooled NAT testing in a Pooled NAT testing in a blood donor settingblood donor setting
24 x negativeresults
Re-test ALLIndividual samples
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Residual risk and NATResidual risk and NAT
BBVBBV PrePre--NAT NAT (<1997)(<1997)
PostPost--NATNAT(>2000)(>2000)
HIVHIV 1 in 1,250,0001 in 1,250,000 1 in 4,800,0001 in 4,800,000
HCVHCV 1 in 230,0001 in 230,000 1 in 3,100,0001 in 3,100,000
HBVHBV 1 in 150,0001 in 150,000 1 in 1,000,0001 in 1,000,000
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
High risk screeningHigh risk screening
6 x negativeresults
Re-test ALLIndividual samples
? Utility in high risk collection centres and high case load primary care centres? Ampliscreen HIV test v1.5HIV Ag-Ab combo test negative samples pooled and NAT tested every 3 days
Cohen M, Busch M etal South Carolina and San Fransisco
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
POC testing POC testing –– rapid testsrapid tests
Use of rapid HIV testing strategiesUse of rapid HIV testing strategiesHigh sensitivity >99%High specificity >99%Good reproducibility †
Unprocessed sample type – capillary bloodLittle laboratory equipment requiredNo need for constant water / electricity supplyFew steps – rapid to performVisual interpretationStorage at room temperature
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Predictive values for rapid HIV testsPredictive values for rapid HIV tests
HIV HIV prevalenceprevalence
0.1%0.1% 1%1% 5%5% 10%10% 30%30%
NPV single NPV single testtest
100%100% 100%100% 99.9%99.9% 99.9%99.9% 99.6%99.6%
PPV single PPV single testtest
9%9% 50%50% 84%84% 92%92% 98%98%
PPV two PPV two teststests
91%91% 99%99% 99.8%99.8% 99.9%99.9% 100%100%
WHO/CDC rapid testing guidelines 2004WHO/CDC rapid testing guidelines 2004
Assuming 99% sensitivity and 99% specificityAssuming 99% sensitivity and 99% specificity
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Rapid testing strategyRapid testing strategy
Blood Sample
Test 1
Test 2
Negative
Result given
Reactive
Discordant
Retest after 6 weeksReferral lab
Positive result
Retest to confirmor Result given
Negative
Result given
Test 2 – may be ELISA or PA
WHO/CDC rapid testing guidelines 2004
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
POC testing issuesPOC testing issues
Regulatory / licensing issuesRegulatory / licensing issuesEnvironmental factorsEnvironmental factorsTraining Training Test counseling Test counseling Performance characteristicsPerformance characteristicsCostCostUtility in certain settingsUtility in certain settings
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Monitoring effect of ARV TherapyMonitoring effect of ARV Therapy
viral loadviral loadTest of choice
when to start therapywhen to change therapyrelative benefit of different regimens
CD4+ lymphocyte countCD4+ lymphocyte countclinical examinationclinical examinationdrug resistance testingdrug resistance testing
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Monitoring effect of ARV TherapyMonitoring effect of ARV Therapy
goals of therapygoals of therapyviral load BLD (50 or 400 copies)viral load BLD (50 or 400 copies)
also clinical benefit survivalVL < 5000 copies
CD4 > 300CD4 > 300--500500prevent resistance prevent resistance
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Quantitative Viral Load AssaysQuantitative Viral Load Assays
Widely available commercial tests include:Widely available commercial tests include:Roche Amplicor HIV-1 Monitor® (PCR)Bayer Quantiplex HIV-RNA (bDNA)Nuclisens nucleic acid sequence base amplification (NASBA)
Emerging new testsEmerging new testsReal time systems – advantages – extended analytical rangeEasyQ - BiomerieuxTaqMan - RocheRealTime - Abbott
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
HIV viral load testsHIV viral load testsManufacturer Principle Results Availability Analytical range
Roche RT-PCR (gag)(COBAS HIV MONITOR v1.5)
copies/mL(6 hours to result)
Widely <50 – 100,000<400 – 750,000
Bayer HIV Branched DNA 3.0 (bDNA) (pol)
copies./mL(results 2x less than Roche)
(36 hours to result)
NSW, Vic <50 – 800,000
Biomerieux HIV-1 QT NASBA (gag)
Copies/mL(6 hours to result)
NSW <400 – 1,000,000<80 – 500,000
Roche Real time (Taqman)(gag)
Copies/mL(4-6 hours to result)
New (no sites)
<40 – 10,000,000
Biomerieux EasyQ HIV-1 real time TMA (gag)
Copies/mL & IU/mL(4-5 hours to results)
New (no sites)
<40 – 10,000,000
Abbott Celera RealtimePCR m2000 (polintegrase)
copies/mL Evaluation <40 – 10,000,000
Artus Realtime PCR –Rotorgene
Copies/mL Evaluation <40 – 10,000,000
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Interpretation of VL ResultsInterpretation of VL Results
different viral strains different viral strains -- subtypessubtypesday to day variabilityday to day variabilityinterinter--laboratory variabilitylaboratory variabilityvariability between assaysvariability between assaysinterinter--current infectionscurrent infectionsrecent vaccinationrecent vaccinationspecimen quality specimen quality -- transport laboratory factorstransport laboratory factorsbaseline baseline -- 2 x tests 2 x tests -- 4 weeks apart4 weeks apart
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
VLT Assay variabilityVLT Assay variabilityPCR is exponential PCR is exponential –– log transform copy numberslog transform copy numbers0.25 log0.25 log(10)(10) variation is normal variation is normal (between labs same method)(between labs same method)
5.95889,1405.45288,1205.70500,000
5.03106,7004.5233,7404.7860,000
4.2517,8003.755,6004.0010,000
3.031,0702.533402.78600
1.95881.45301.7050
HIVHIV--1 RNA copy1 RNA copy Acceptable differenceAcceptable difference
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
VLT Assay variabilityVLT Assay variabilityPCR is exponential PCR is exponential –– log transform copy numberslog transform copy numbers0.20 log0.20 log(10)(10) variation is normalvariation is normal (within lab)(within lab)
5.90794,3305.50316,2305.70500,000
4.9895,5004.5838,0204.7860,000
4.2015,8503.806,3104.0010,000
2.989542.583802.78600
1.90791.50321.7050
HIVHIV--1 RNA copy1 RNA copy Acceptable differenceAcceptable difference
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Undetectable by the most sensitive testUndetectable by the most sensitive testH
IV R
NA
(cop
ies/
mL)
10,000
1,000
100
10
1
100,000
0 10 20 30 40 50 60 70
LLD Standard VLT
LLD Ultrasensitive
Weeks on Therapy
20 weeks earlier
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Recommendations for resistance Recommendations for resistance testingtesting
1.1. Treatment naTreatment naïïve patients with acute or recent ve patients with acute or recent infection infection virologicvirologic failure during therapyfailure during therapy
2.2. Therapy failure including suboptimal viral Therapy failure including suboptimal viral suppression after initiation of ARV suppression after initiation of ARV
3.3. Pregnant HIV infected women and Pregnant HIV infected women and paediatricpaediatric cases cases with detectable virus load, when therapy change is with detectable virus load, when therapy change is consideredconsidered
4.4. ‘‘sourcesource’’ patient when post exposure prophylaxis is patient when post exposure prophylaxis is consideredconsidered
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
ConsiderationsConsiderations
drug nadrug naïïve patients with chronic infection in ve patients with chronic infection in who treatment is to be startedwho treatment is to be started
Recommend testing the earliest stored sample if suspicion of resistance is high or prevalence of resistance in the population exceeds 10%
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
How is it done?How is it done?
Genotypic resistanceGenotypic resistanceDNA sequencing – mutation detectionInterpretation issues - consensusSubtype determination possible$200 - $400
Phenotypic resistancePhenotypic resistanceVirus exposed to drugMolecular based methods>$1000
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
Breakthrough of HIV Breakthrough of HIV resistanceresistance
NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006
DNA sequencing and Virtual Phenotype
-Computer assisted interpretation-Issues with update in rapidly evolving virus-Online updates-In house sequencing advantages